1. Field of the Invention
The present invention concerns at least the fields of cell biology, molecular biology and medicine. In particular aspects, the present invention concerns the fields of treatment and/or prevention of prostate cancer.
2. Related Art
Prostate cancer is the most commonly diagnosed malignancy in males in the United States and the second leading cause of male cancer mortality. In the event that surgery and/or radiation do not cure prostate cancer, systemic therapy is based on inhibiting the androgen receptor (AR). The androgen receptor is a steroid receptor transcription factor which promotes the growth and survival of normal and cancerous prostate cells. Androgen ablation is used to block the activation or activity of androgens initially and results in a favorable clinical response. However, prostate cancer invariably occurs in a manifestation which is fatal and is resistant to androgen ablation. This stage of prostate cancer is termed androgen refractory prostate cancer.
Prostate cancer is the second leading cause of cancer death among men in western countries. Patients with advanced prostate cancer initially benefit from androgen ablation therapy which leads to temporary remission of the tumor due to apoptosis of androgen-sensitive tumor cells. However, the recurrence of androgen independent tumors is inevitable for most patients and renders the conventional hormone therapy ineffective (Denmeade, S. R. et al., Nat Rev Cancer 2, 389-96 (2002)). It has, therefore, become a focus of intensive study to understand the mechanisms underlying progression of hormone refractory prostate cancer (Litvinov, I. V., et al., Prostate 61, 299-304 (2004); Feldman, B et al., Nat Rev Cancer 1, 34-45 (2001); and Debes, J. D. et al., N Engl J Med 351, 1488-90 (2004)). Development of new effective therapeutic agents is necessary for targeting hormone refractory prostate cancer. As prostate cancer is a product of slow but continuous accumulation of altered genes and proteins controlling several signaling pathways it thus poses a difficult task for identification of precise markers to predict high risk for recurrence. Validation of tumor progression markers are an essential component for prostate cancer treatment strategies to plan for appropriate therapy based on its level, tissue distribution and the stage or extent of the disease.
Identification and characterization of the molecular mechanisms and biological switches which propel normal prostate cells into cells in the cancerous state is critical for developing therapeutic modalities which can be used to prevent and/or treat male subjects predisposed to prostate cancer.
Increasing clinical findings demonstrate that a majority of androgen ablation therapy-resistant prostate cancers still express AR and androgen-dependent genes, indicating that the AR-signaling pathway is functional in the absence of androgens or in the presence of low levels of androgens (Chang, C. S. et al., Science 240, 324-6 (1988); Lubahn, D. B. et al., Mol Endocrinol 2, 1265-75 (1988)). Several independent studies also showed that AR is essential for both hormone sensitive and recurrent hormone refractory prostate cancer (McPhaul, M. J. et al., J Investig Dermatol Symp Proc 8, 1-5 (2003); Heinlein, C. A. et al., Endocr Rev 25, 276-308 (2004)). The mechanisms underlying androgen-independent activation of AR have yet to be fully understood. Mutations and amplification of AR, alterations in protein kinases, growth factors and nuclear receptor coactivators have all been proposed to modulate AR signaling and may, therefore, play key roles in the development of androgen independence of prostate cancer (Feldman, B. et al., Nat Rev Cancer 1, 34-45 (2001); Lubahn, D. B. et al. Mol Endocrinol 2, 1265-75 (1988); Kuiper, G. G. et al., J Mol Endocrinol 2, R1-4 (1989)). Mutations in the ligand binding domain of AR are shown to broaden the ligand binding profile of the mutant receptor (Lubahn, D. B. et al., Proc Natl Acad Sci USA 86, 9534-8 (1989); Libertini, S. J. et al., Cancer Res 67, 900 1-5 (2007); Simental, J. A., et al., J Biol Chem 266, 5 10-8 (1991)). However, the frequency of AR mutation is generally low and probably only accounts for less than 10% of the cases surveyed (Jenster, G. et al. Mol Endocrinol 5, 1396-404 (1991)). Upregulation of the enzymes involving in steroid synthesis in some recurrent prostate tumors and activation of AR via the intracrine mechanism have also been reported (Rundlett, S. E. et al., Mol Endocrinol 4, 708-14 (1990)). However, the tissue androgen level did not correlate with clinical prognosis. Recently, the increased AR expression level is shown to associate with the development of resistance to anti-androgen therapy (McPhaul, M. J. et al., J Investig Dermatol Symp Proc 8, 1-5 (2003)). The cross-talk between growth factor and AR signaling pathways in prostate cancer cells has been well documented. The expression of several peptide growth factors, such as EGF/TGFa, IL-6 and IGF-1, are reported to be elevated during progression to hormone refractory human prostate cancer (Bolton, E. C. et al., Genes Dev 21, 2005-17 (2007); Wang, Q. et al., Mol Cell 27, 3 80-92 (2007); Clegg, N. et al. J Steroid Biochem Mol Biol 80, 13-23 (2002); DePrimo, S. E. et al., Genome Biol 3, RESEARCH0032 (2002); Marcelli, M. et al., J Clin Endocrinol Metab 84, 3463-8 (1999); Comuzzi, B. et al., Am J Pathol 162, 233-41 (2003); Chen, C. D. et al., Nat Med 10, 33-9 (2004)). These autocrine/paracrine factors can either induce the androgen-independent activation of AR transcriptional activity or sensitize AR to low concentrations of androgens (Lubahn, D. B. et al., Mol Endocrinol 2, 1265-75 (1988); Zegarra-Moro, O. L., et al., Cancer Res 62, 1008-13 (2002); Culig, Z. et al., Endocr Relat Cancer 9, 155-70 (2002)). Several protein kinases, including MAPK, Akt/PKB, PICA and PKC, Src and Ack-1 have been shown to modulate AR transcriptional activity by phosphorylating AR or its coactivators such as TIF2 and SRC1 (Zegarra-Moro, O. L., et al., Cancer Res 62, 1008-13 (2002); Culig, Z., et al., Endocr Relat Cancer 9, 155-70 (2002); Gelmann, E. P. et al., J Clin Oncol 20, 3001-15 (2002); Mizokami, A. et al., Cancer Res 64, 765-71 (2004); Kurita, T. et al., Cell Death Differ 8, 192-200 (2001); Veldscholte, J. et al., Biochem Biophys Res Commun 173, 534-40 (1990); Taplin, M.-E. et al., J Clin Oncol 21, 2673-2678 (2003)). Another plausible hypothesis for activation of androgen receptor activity in the absence of hormones was proposed by Tepper et al. who showed that a mutant AR identified in hormone refractory prostate cancer cell line CWR22Rv1 contains an in-frame tandem duplication of exon 3 that encodes the second zinc finger of the AR DNA-binding domain (Paez, J. G. et al., Science 304, 1497-1500 (2004)). This insertional mutation renders AR susceptible to the protease cleavage at the hinge region and generates a constitutively active form around 80 kD. A recent study also showed that calpain may mediate cleavage of the AR mutant in this cell line (Yeh, S. et al., Proc Natl Acad Sci USA 96, 5458-63 (1999)). However, the frequency of such insertional mutation has only been detected in CWR22R xenograft derived cell line CWR22Rv1. It remains elusive whether it is a general mechanism underlying androgen-independence.
We have cloned novel AR splice variants (AR3, AR4, AR4b, AR5 and AR8) from hormone refractory CWR22Rv1 xenografts. All the variants contain the intact N-terminal transactivation domain and the DNA binding domain, but lack the ligand binding domain, and therefore, are true androgen-independent. AR3 appears to be constitutively active in ARE-mediated transcription. We confirmed the expression of these AR variants in human prostate tumors by RT-PCR and/or immunohistochemistry. Knocking-down AR3, one of the major AR variants in hormone refractory prostate cancer cells, attenuated androgen-independent growth in both cell culture and xenograft models. Our data suggest that the AR variants resulted from alternative splicing may be a novel mechanism underlying androgen-independence during prostate cancer progression. These novel AR variants are not inhibited by currently available anti-androgen drugs (such as casodex) due to the lack of the ligand binding domain. Therefore new drugs targeting at these novel AR variants may potentially be more effective for androgen-independent prostate tumors. Further characterization of these AR variants will provide new insights into mechanisms underlying androgen-independence and identify new therapeutic targets. Detection of the expression of these AR variants may be potentially used as a prognostic marker for prostate cancer and targeting these variants may develop more effective treatment for androgen-independent prostate cancer.